It is an urgent problem to establish disease prevention and therapeutic methods including infection prevention technology for the mammals including human (specifically domestic animals, pet animals, etc.), birds (specifically farmed chicken, pet birds, etc.), amphibian animals, reptiles, fish (specifically aqua cultured fish, pet fish, etc.) and invertebrates. Furthermore, in order to achieve this, the methods using no chemicals, without environmental pollution, without producing resistant bacteria and without accumulation in the human body are strongly required. The present inventors have already found for the above problems that the immunopotentiators derived from plants, such as water extract of wheat safely achieve the disease prevention and therapeutic effects (Patent document 1, Non-patent document 1). Also in order to achieve the above object, the present inventors have found that it is possible to use low molecular weight lipopolysaccharides obtained from Pantoea agglomerans which is a symbiotic bacterium with wheat (Non-patent document 2). Meanwhile, recent studies have demonstrated that various substances in addition to lipopolysaccharides exhibit the immunopotentiation effect, and these plural natural materials containing the immunopotentiator have attracted attention.
Fermentation technology using bacteria has been commonly used not only in food fields but also broad fields. The fermentation has been widely utilized for the production of alcohols including wines, the production of soy sauces and soybean pastes, the production of fermented milk products such as cheeses, and the production of pharmaceuticals. The bacteria used for these fermentations are many, and rice malt (fungus) yeast and lactic acid bacteria are representative, but it has been rarely reported to use gram-negative bacteria. In general, the fermentation is a phenomenon that organic matter is decomposed by an action of the bacteria, and means in the broad sense that a useful substance is produced by the bacteria (Non-patent document 3). Representatives of the fermentation using the bacteria include wine-making. The wine-making is the fermentation technology using wine yeast adhering to the fruit skin of grapes, and its product is alcohol. In the fermentation technology using the bacteria, as those using gram-negative bacteria, methane fermentation using methane bacteria, acetic fermentation using acetic bacteria and ethanol fermentation (tequila fermentation) from rootstocks of maguey using Zymomonas mobilis have been known, but fermentation culture using an edible plant as a material and using the bacteria characterized by living in a symbiotic relationship with the plant have been rarely known, and the immunopotentiator has never attracted attention as a fermented product. Still more, the method for fermentation and culture for the purpose of producing the immunopotentiator has never attracted attention.
Meanwhile, when the fermentation is performed by the bacteria, generally there are nutrient conditions which a fermentation substrate should meet for bacteria growth. That is, the presence of substances available as nutrients by the bacteria is essential, i.e., monosaccharides such as glucose and fructose as carbon sources are sufficiently contained. Therefore, fruits such as grapes containing abundant fructose can be utilized as the fermentation substrate without giving any processing. However, in other cases, a pretreatment such as heating and enzyme treatment for the fermentation by the bacteria is required. For example, the foregoing Zymomonas mobilis is a bacterium used for the tequila fermentation. In this case, polysaccharides obtained from the rootstocks of the maguey which is not edible plant are decomposed into fermentable monosaccharides by heating, and subsequently the monosaccharides are fermented by the bacteria to yield the alcohol as the fermentation product. Therefore, when the fermentation culture is performed using a typical bacterium, the polysaccharides such as starch are not suitable as the fermentation substrate. For example, it has been described that Pantoea agglomerans cannot decompose starch (Non-patent document 4).
We have demonstrated that an active component for potentiating the immunity is contained in an aqueous extract of wheat flour (Non-patent document 5). We have also demonstrated that the active components are contained in food grains (wheat, rice), seaweeds (brown seaweed, kelp, hijiki (brown alga) and layer) and beans (soybean and adzuki bean) (Non-patent document 6). As this biological activity, it has been found to have preventive effects on human and mouse diseases (diabetes, hyperlipemia, atopic dermatitis, cancer) and can be effective for infection prevention of fish, crustacea and chickens (Patent document 1, Non-patent document 1). However, to expect the above effect by the aqueous extract of wheat flour, it is necessary to ingest the wheat flour in a large amount.
Meanwhile, Pantoea agglomerans is a bacterium which lives in a symbiotic relationship with wheat, and is considered to be useful in wheat cultivation because the bacterium supplies phosphorus and nitrogen to the wheat (Non-patent document 7). Also, Pantoea agglomerans deposits not only on wheat but also on epidermis of pears and apples. It has been demonstrated in Europe that rot diseases due to fungi can be prevented when this bacterium deposits, and development of utilizing this bacterium as an environmentally friendly fungicide with no toxicity has been advanced (Non-patent document 8). It has been defined that symbiosis is “a phenomenon in which xenogeneic organisms live together. In this case, it is common to mean constantly keeping a behaviorally or physiologically close relationship. Therefore, it does not fall into this concept only to live in the same habitat. Symbiosis is classified and divided into various categories depending on the life meaning and essentiality of the symbiotic partner, sustainability of the relationship and spatial positioning of the symbiotic partner. Generally, the symbiosis is broadly divided into three, mutualism, commensalism and parasitism on the basis of the presence or absence of life benefit/disbenefit of the symbiotic partners.” (Non-patent document 9). It has been known that Pantoea agglomerans is separated from wheat in any regions and any types (Non-patent document 5) and also separated from fruits (Non-patent documents 10, 11). It has been reported that Pantoea agglomerans protects plants from fungi or other bacteria by producing antibiotics (Non-patent documents 12, 13) and performs phosphorus and nitrogen fixation (Non-patent document 7). Therefore, it is considered that Pantoea agglomerans is always present in plants and plays a role to give benefits to plants. Thus, its living mode is regarded as “symbiosis” but not “parasitism”. In addition, we have demonstrated that the active component to potentiate the immunity is contained in Pantoea agglomerans. Also, we have found that the low molecular weight lipopolysaccharide obtained from this bacterium has preventive effects on human and mouse diseases (diabetes, hyperlipemia, atopic dermatitis, cancer) and is effective for infection prevention of fish, crustacea and chickens (Patent document 3, Non-patent document 2).
In such a circumstance, we have conceived the idea of establishing a method for producing a fermented plant extract using Pantoea agglomerans as a method for producing a safe and inexpensive immunopotentiator. That is, we have focused on (1) culturing Pantoea agglomerans at low cost using a medium containing major protein components included in a culture solution derived from plants as well as fermenting a plant component and (2) preparing materials abundantly containing Pantoea agglomerans contained in the plant or a product by fermentation, thereby developing pharmaceuticals, pharmaceuticals for animals, quasi drugs, cosmetics, functional foods, foods, feedstuff and bath agents, for mammals including humans (specifically domestic animals, pet animals, etc.), birds (specifically farmed chicken, pet birds, etc.), amphibian animals, reptiles, fish (specifically aqua cultured fish, pet fish, etc.) and invertebrates. However, this does not mean that the bacteria living in a symbiotic relationship with a plant can directly utilize the plant components, e.g., the material derived from an edible plant as a fermentation substrate. For example, wheat flour is a composite organic substance of starch and the like present in wheat grains, but isolated from Pantoea agglomerans which is a symbiotic bacterium with wheat via the outer skin, and does not directly make contact. Thus, it cannot be demonstrated by a symbiotic relationship of the bacteria with wheat so as to whether Pantoea agglomerans can ferment and be cultured using wheat flour or not. In fact, it has not been known and reported at all that Pantoea agglomerans can assimilate wheat flour. Conversely, on the basis of publicly known facts, it has been described that Pantoea agglomerans cannot utilize wheat starch as the fermentation substrate.
Glucides contained in plants are often retained as starch, and this is remarkable in edible plants, particularly food grains. Usually, bacteria do not have a function in which starch is highly assimilated. In this regard, it has been known that a part of facultative gram-negative bacteria can ferment starch. For example, Erwinia is known to be able to assimilate starch. However, in this fermentation, when starch is fermented, it is intended to utilize an amylase activity of the bacteria by adding the bacteria cultured in a large amount in another optimal medium, and it has never been conceived that the culture itself is performed using starch and fermentation is performed in conjunction therewith. In the conventional technology, it is regarded as the objective fermentation to only effectively utilize the amylase activity of the bacteria, and it is not scheduled to grow the bacteria using starch as the substrate. Meanwhile, in the Examples of the present invention, it is disclosed that a fermented product is produced in addition to the growth of the bacteria by using starch as an only carbon source, and the present invention is significantly different from the conventional technology in that the present Example is not only fermentation but also fermentation and culture.
On the other hand, if a certain bacterium retains the function to decompose starch, this does not directly mean that the bacteria can grow using starch as the substrate. Upon the culture, in the case of also aiming at the growth of the bacteria, the amount of the bacteria added at the start of the culture is extremely small. In such a case, even if the bacterium lightly has amylase activity, this activity is too weak to sufficiently decompose the substrate and the growth of the bacterium is not achieved. In fact, it has been considered that many of the bacteria cannot grow using starch as the only carbon source.
However, if the fermentation and culture can be performed using Pantoea Agglomerans in the medium containing wheat flour as a major component to produce a fermented plant extract (hereinafter, the fermented plant extract obtained by fermentation and culture using Pantoea Agglomerans in the medium containing wheat flour as the major component is referred to as a fermented wheat extract) abundantly containing an immunopotentiator at low cost, as specific examples, pharmaceuticals, pharmaceuticals for animals, quasi drugs, cosmetics, foods, functional foods, feedstuff, and bath agents which are environmentally friendly, safe and effective for infection prevention for humans and in the fields of animal industry and aqua culture should be able to be provided. The present invention has been completed by taking the opportunity that it was discovered that Pantoea Agglomerans grew using wheat flour as the substrate in the above context and by extensively conducting many experiments.
The fermented plant extract provided by the present invention is a generic term which includes a culture solution itself obtained by performing fermentation and culture, a liquid component obtained by solid/liquid separation of this culture solution, and a liquid component obtained by giving an extraction process to a solid component obtained by the solid/liquid separation, and the like. That is, the fermented plant extract includes the culture solution itself obtained by the method for fermentation and culture according to the present invention, and all extracts capable of being prepared using a whole or a part of the culture solution. Although it is as a matter of course, the fermented plant extract can be utilized by drying as fermented plant extract powder or dissolving the fermented plant extract powder at an optional concentration in an appropriate solution, e.g., phosphate buffer solution including normal saline solution.    [Patent document 1] Japanese Unexamined Patent Application Publication No. H3-218466    [Patent document 2] Japanese Unexamined Patent Application Publication No. H8-198902    [Patent document 3] WO 00/57719    [Patent document 4] Japanese Unexamined Patent Application Publication No. H6-78756    [Patent document 5] Japanese Unexamined Patent Application Publication No. H4-187640    [Patent document 6] Japanese Unexamined Patent Application Publication No. H4-49240    [Patent document 7] Japanese Unexamined Patent Application Publication No. H4-99481    [Patent document 8] Japanese Unexamined Patent Application Publication No. H5-155778    [Non-patent document 1] Inagawa, H. et al., Biotherapy 5(4), p 617-621, 1991    [Non-patent document 2] Soma G. et al., “Tumor necrosis Factor: Molecular and Cellular Biology and Clinical Relevance” p 203-220, 1993    [Non-patent document 3] Yamada T. et al., “Seibutsugaku Jiten” 3rd ed., p 1021, 1983    [Non-patent document 4] Gavini, F. et al., Int. J. Syst. Bacteriol., 39, p 337-345, 1989    [Non-patent document 5] Nishizawa T. et al., Chem. Pharm. Bull., 40(2), p 479-483, 1992    [Non-patent document 6] Inagawa H. et al., Chem. Pharm. Bull., 40(4), p 994-997, 1992    [Non-patent document 7] Neilson A. H., J. Appl. Bacteriol., 46(3), p 483-491, 1979    [Non-patent document 8] Nunes C. et al., Int. J. Food Microbiol., 70(1-2), p 53-61, 2001    [Non-patent document 9] Yamada T. et al., “Seibutsugaku Jiten” 3rd ed., p 287-288, 1983    [Non-patent document 10] Nunes C. et al., J. Appl. Microbiol., 92(2), p 247-255, 2002    [Non-patent document 11] Asis C. A. Jr. et al., Lett. Appl. Microbial., 38(1), p 19-23, 2004    [Non-patent document 12] Vanneste J. L. et al., J. Bacteriol., 174(9), p 2785-2796, 1992    [Non-patent document 13] Kearns L. P. et al., Appl. Environ. Microbiol., 64(5), p 1837-1844, 1998